Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

Abstract

Blockade of the inhibitory PD-1/PD-L1 immune checkpoint axis is a promising cancer treatment. Nonetheless, a significant number of patients and malignancies do not respond to this therapy. To develop a screen for response to PD-1/PD-L1 inhibition, it is critical to develop a non-invasive tool to accurately assess dynamic immune checkpoint expression. Here we evaluated non-invasive SPECT/CT imaging of PD-L1 expression, in murine tumor models with varying PD-L1 expression, using high affinity PD-L1-specific nanobodies (Nbs). We generated and characterized 37 Nbs recognizing mouse PD-L1. Among those, four Nbs C3, C7, E2 and E4 were selected and evaluated for preclinical imaging of PD-L1 in syngeneic mice. We performed SPECT/CT imaging in wild type versus PD-L1 knock-out mice, using Technetium-99m (99mTc) labeled Nbs. Nb C3 and E2 showed specific antigen binding and beneficial biodistribution. Through the use of CRISPR/Cas9 PD-L1 knock-out TC-1 lung epithelial cell lines, we demonstrate that SPECT/CT imaging using Nb C3 and E2 identifies PD-L1 expressing tumors, but not PD-L1 non-expressing tumors, thereby confirming the diagnostic potential of the selected Nbs. In conclusion, these data show that Nbs C3 and E2 can be used to non-invasively image PD-L1 levels in the tumor, with the strength of the signal correlating with PD-L1 levels. These findings warrant further research into the use of Nbs as a tool to image inhibitory signals in the tumor environment.

Keywords: SPECT/CT imaging; biomarker; immune checkpoints; nanobodies; programmed death-1/programmed death-Ligand 1.

Figure 1. Selection of anti-mouse-PD-L1 specific Nbs

(A) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. (B) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown (n = 1). (C) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 (n = 3).

Figure 2. SPECT/CT and biodistribution evaluated 1 hour after injection of ^99mTc-Nbs C3, C7, E4 or E2 in naive wild type (WT) and PD-L1 knock out (KO) mice

(A) Results of SPECT/CT scans to determine the biodistribution of ^99mTc-Nbs C3, C7, E4 or E2 injected in WT or PD-L1 KO mice (n = 3). (B) Gamma counting of isolated organs from WT or KO mice injected with ^99mTc-Nbs C3, C7, E4 or E2. The graph summarizes the organ uptake of the Nb per gram organ as the mean ± SEM (n = 3). (C) Percentage PD-L1 positive cells in spleen, lymph node (LN) and brown adipocyte tissue (BAT) of WT and PD-L1 KO mice using flow cytometry. The graph summarizes the percentage PD-L1 positive cells as mean ± SEM (n = 3). K = kidney, L = liver, S = spleen, B = bladder, BAT = brown adipocyte tissue.

Figure 3. SPECT/CT in the shRNA-modified TC-1 model 1 hour after injection of ^99mTc-Nbs C3 or E2

(A) Percentage of PD-L1 on TC-1 cells transduced with lentiviral vectors harboring shRNA against mouse PD-L1 (knock-down, KD), wild type TC-1 cells (WT) or TC-1 cells transduced with mouse PD-L1 (knock-in, KI), evaluated with flow cytometry (n = 5). The graph summarizes the percentage PD-L1 positive cells as mean ± SEM. (B) TC-1 KD or KI cells were injected subcutaneously at the tail base of C57BL/6 mice. Tumor growth was followed every other day. The evolution of tumor size is shown as mean ± SEM (n = 5). (C) Mice were sacrificed on day 12 and tumors were isolated, after which expression of PD-L1 on tumor cells (CD45⁻, white bar) and tumor-infiltrating immune cells (CD45⁺, black bar) was evaluated in flow cytometry. The graph summarizes the percentage of PD-L1 as mean ± SEM (n = 5). (D) Images of SPECT/CT scans to determine the accumulation of ^99mTc-Nbs C3 and E2 in C57BL/6 mice bearing KI (left panel) or KD (right panel) tumors (n = 6). The red arrow indicates the tumor on the images. (E) Graphs showing the quantified ex vivo analysis to determine the accumulation of ^99mTc-Nbs C3 and E2 in PD-L1 KI (black bar) or KD (white bar) tumors. The graph shows the percentage radioactivity per gram tumor as mean ± SEM (n = 6).

Figure 4. Use of the CRISPR/Cas9 technology to generate a PD-L1 KO tumor model

(A) Percentage of PD-L1 as assessed with flow cytometry on TC-1 cells transduced with lentiviral vectors harboring CRISPR/Cas9 targeted to mouse PD-L1 (knock-out, KO) compared to WT and KD TC-1 cells either or not pre-treated with recombinant mouse IFN-γ (50 ng/mL) (n = 3). The graph summarizes the percentage PD-L1 positive cells as mean ± SEM. (B) TC-1 KO or WT cells were injected subcutaneously at the tail base of WT or PD-L1 KO mice. Tumor growth was followed every other day. The tumor size in function of time is shown as mean ± SEM (n = 3). (C) TC-1 KO cells were injected subcutaneously at the tail base of PD-L1 KO mice, which were pretreated with a CD8⁺ depleting antibody or an isotype matched control antibody. The percentage CD8 positive cells in the blood was evaluated using flow cytometry. The graph shows a representative histogram of CD8 positive cells in mice pretreated with an isotype matched control antibody (grey line) or a CD8 depleting antibody (red line). (D) TC-1 KO cells were injected subcutaneously at the tail base of PD-L1 KO mice, which were pretreated with a CD8⁺ depleting antibody or an isotype matched control antibody. WT TC-1 cells were injected subcutaneously at the tail base of WT mice. Tumor growth was followed every other day. The tumor size in function of time is shown as mean ± SEM (n = 3).

Figure 5. SPECT/CT in the CRISPR/Cas9-modified TC-1 model 1 hour after injection of ^99mTc-Nbs C3 or E2

(A) Images of the SPECT/CT scans to evaluate ^99m Tc-Nbs C3 and E2 for tumor stratification in CD8-depleted PD-L1 KO mice bearing PD-L1 KO tumors (KO) or WT mice bearing WT (PD-L1⁺) tumors (WT) (n = 6). The red arrow indicates the tumor on the images. (B) CD8-depleted PD-L1 KO mice injected with PD-L1 KO tumors or WT mice injected with WT (PD-L1⁺) TC-1 cells were sacrificed on day 17 and tumors were isolated, after which expression of PD-L1 on cancer cells (CD45⁻, white bar) and tumor-infiltrating immune cells (CD45⁺, black bar) was evaluated in flow cytometry. The graph summarizes the percentage of PD-L1 as mean ± SEM (n = 6). (C) Results of the gamma counting of isolated organs from CD8-depleted PD-L1 KO mice bearing PD-L1 KO tumors (KO, white bars) or WT mice bearing WT (PD-L1⁺) tumors (WT, black bars) injected with ^99Tm Tc-Nbs C3 and E2. The graph summarizes the %IA/g as mean ± SEM (n = 6). (D) Tumor uptake (%ID/cc) calculated via ROI analysis on the periphery of the tumor from CD8-depleted PD-L1 KO mice bearing PD-L1 KO tumors (KO, white bars) or WT mice bearing PD-L1⁺ tumors (WT, black bars) injected with ^99Tm Tc-Nbs C3 and E2. The graph summarizes the %ID/cc as mean ± SEM (n = 6).